In gene analysis, radioisotopes have heretofore been used as a modifier for labeling a nucleotide chain such as DNA, RNA, oligonucleotides, or nucleic acids. However, these radioisotopes have a limited duration attributed to half-life, the limited number of handling facilities, radiation exposure, difficulty of discarding, etc. Therefore, the use thereof is on a downward trend. In recent years, modifiers such as fluorescent materials (e.g., fluorescein) and biotin have been used generally as a substitute for the radioisotopes.
Methods of modifying the 5′-terminus of a nucleotide chain via the phosphate group have been proposed. These methods involve modifying the 5′-terminus of the nucleotide chain with a modifier through a chemical cross-linking condensation reaction using the 5′-terminal phosphate group as a functional group (e.g., Non-Patent Documents 1 to 2 (listed later; the same holds true for the description below). Alternatively, methods of modifying a nucleotide chain during chemical synthesis thereof have also been proposed. These methods involve introducing a phosphate group labeled with a modifier into the nucleotide chain during the chemical synthesis (e.g., Patent Documents 1 to 4 and Non-Patent Documents 3 to 4). The methods can also be applied to the automatic chemical synthesis of nucleotide chains and have therefore been used frequently.
However, the methods of modifying the 5′-terminus are capable of modifying one nucleotide chain molecule with only one modifier and also require a long time for the cross-linking condensation reaction. The methods of modifying a nucleotide chain during the chemical synthesis thereof allow for chemical synthesis of allegedly up to 130 nucleotides. The synthesis method thereof adds and polymerizes the nucleotides one by one and has addition/polymerization efficiency of up to 99% in a single run. Therefore, the synthesis of a larger number of nucleotides results in lower synthesis yields. To acquire sufficient yields, the number of nucleotides must be limited (e.g., Non-Patent Document 5). Furthermore, a color or luminescent reaction using alkaline phosphatase has been used frequently in gene detection by virtue of the high sensitivity thereof (e.g., Non-Patent Documents 6 to 7). However, the alkaline phosphatase has the property of dephosphorylating the 5′-terminus of the nucleotide chain. Therefore, the modifier is dissociated from the nucleotide chain. Thus, the alkaline phosphatase cannot be used for the nucleotide chain having the modified 5′-terminus.
In addition to the methods of modifying the 5′-terminus, methods using a replication or transcription reaction have also been proposed. These methods involve incorporating a nucleotide modified in advance with a modifier into a nucleotide chain through the replication or transcription reaction to obtain the nucleotide chain modified with the modifier. The replication reaction is performed by, for example, nick translation, random primer, and primer extension methods previously used in radioisotope labeling. In this case, deoxyribonucleotide 5′-triphosphate modified in advance with a modifier instead of radioisotopes is incorporated into the nucleotide chain using the replication reaction catalyzed by DNA polymerase. Alternatively, ribonucleotide 5′-triphosphate modified in advance with a modifier is incorporated into the nucleotide chain using the transcription reaction catalyzed by RNA polymerase (e.g., Patent Documents 5 to 8 and Non-Patent Documents 6 and 8 to 9). These modification methods advantageously have simplified procedures, a high amount of nucleotide chain modification, modifier stability, and the lack of modifier dissociation caused by alkaline phosphatase and have therefore been used frequently as a method suitable for gene analysis. Furthermore, these modification methods can be used in combination with a polymerase chain reaction (PCR) or RNA amplification reaction to simultaneously achieve gene amplification and labeling reactions (e.g., Non-Patent Documents 10 to 12). Therefore, these methods have been used frequently in comprehensive gene analysis typified by DNA microarrays (e.g., Patent Documents 9 to 10 and Non-Patent Documents 13 to 15).
However, the modification methods using a replication or transcription reaction are based on the incorporation of a nucleotide modified in advance with a modifier, that is, an artificial nucleotide. This artificial nucleotide is inferior in incorporation efficiency to original nucleotides and is also inferior in amplification efficiency to the original nucleotides, even when used in combination with PCR (e.g., Patent Document 8 and Non-Patent Document 16). In addition, the amount of modifiers incorporated is as variable as 12 to 25 molecules per nucleotide chain molecule (e.g., Non-Patent Document 17), leading to unfavorable reproducibility of the amount of modification. Moreover, the modifier incorporated into the nucleotide chain inevitably causes steric hindrance during hybridization, that is, formation of a nucleotide chain duplex, in gene analysis. Furthermore, the synthesis of the nucleotide modified in advance with a modifier is complicated. Not everyone can easily synthesize such a nucleotide (e.g., Non-Patent Documents 8 and 18). As a result, the type of the modifier that can be used is limited.
Other modification methods have also been proposed, which do not require a nucleotide chain serving as a template as required in the modification methods using a replication or transcription reaction. These methods can modify a nucleotide chain through an addition reaction of a nucleotide in the absence of the template. Examples of the methods include: a method of tailing, using terminal deoxynucleotidyl transferase, the 3′-terminus of a nucleotide chain with deoxyribonucleotide 5′-triphosphate or dideoxyribonucleotide 5′-triphosphate modified in advance with a modifier (e.g., Non-Patent Documents 19 to 21); and a method of tailing, using RNA ligase, a nucleotide chain with deoxyribonucleotide-3′,5′-bisphosphate or ribonucleotide-3′,5′-bisphosphate modified in advance with a modifier (e.g., Patent Document 11 and Non-Patent Document 22). Among them, the method of tailing with dideoxyribonucleotide 5′-triphosphate or the method of tailing with ribonucleotide-3′,5′-bisphosphate is capable of modifying one nucleotide chain molecule with only one modifier molecule. By contrast, the method of tailing with deoxyribonucleotide 5′-triphosphate modified in advance with a modifier is capable of modifying one nucleotide chain molecule with plural modifier molecules.
However, such a method of tailing with deoxyribonucleotide 5′-triphosphate modified in advance with a modifier varies in the number of the modified nucleotides added depending on reaction conditions and disadvantageously forms an unnecessary nucleotide sequence. This unnecessary nucleotide sequence is also responsible for a mismatch during hybridization in gene analysis. As also described in the modification methods using a replication or transcription reaction, not everyone can easily synthesize such a nucleotide modified in advance with a modifier. As a result, the type of the modifier that can be used is limited.
A modifier can be introduced into the 3′-terminus of a nucleotide chain not only by the tailing but by chemical synthesis. In the latter case, an unnecessary nucleotide sequence is not formed. However, the unfavorable yields and constraints of a nucleotide chain length already described in the modification methods using chemical synthesis cannot be circumvented.
Nucleotide chain modification is performed not only for labeling but for immobilizing the nucleotide chain onto a substrate. The substrate previously used for immobilization is, for example, a nitrocellulose, nylon, or polyvinylidene fluoride substrate processed to have hydrophobicity and positive charge. In this case, the nucleotide chain is immobilized onto the substrate through a hydrophobic bond between the nucleotide chain and the substrate or through an electrostatic bond between the negatively charged phosphate group of the nucleotide chain and the positively charged substrate.
In this immobilization method, a longer nucleotide chain length results in higher substrate occupancy per nucleotide chain molecule. Therefore, the amount of nucleotide chains immobilized per substrate unit area is reduced. Moreover, the nucleotide chain immobilized on the substrate is disadvantageously dissociated therefrom. To solve these problems, methods have been used, which involve: modifying the terminus of a nucleotide chain with a modifier having a functional group, while forming a binding group for the functional group on a substrate surface; and cross-linking and condensing the functional group in the modifier with the binding group on the substrate surface to immobilize the nucleotide chain onto the substrate surface through a covalent bond (e.g., Patent Documents 12 to 15 and Non-Patent Document 23). These methods can achieve more stable and more firm immobilization of the nucleotide chain than that through the conventional hydrophobic and electrostatic bonds. Examples of functional group-binding group combinations used include amino-carboxyl, amino-isothiocyanate, amino-aldehyde, amino-succinimide, and amino-epoxy groups.
However, the 5′-terminus of the nucleotide chain is modified via the phosphate group with the modifier having a functional group. As described above, such modification via the phosphate group has disadvantages. Specifically, the nucleotide chain is dissociated therefrom by phosphatase. Accordingly, the nucleotide chain thus modified cannot be used in detection through a luminescent reaction. Alternatively, the 3′-terminus of the nucleotide chain is also modified with the modifier having a functional group. In such a case, the constraints of a nucleotide chain length cannot be circumvented for acquiring sufficient yields that can be synthesized chemically.
To solve these problems associated with nucleotide chain labeling or immobilization, a method of directly modifying the 3′-terminus of a nucleotide chain with a modifier has been proposed (see Patent Document 16). In this method, two or more nucleotides having an uracil base are added to the 3′-terminus of the nucleotide chain; the added nucleotide is degraded with uracil-DNA glycosidase to form an aldehyde group at the 3′-terminus; and the 3′-terminus of the nucleotide chain is directly modified with a modifier having an amino group through a covalent bond between the aldehyde group and the amino group.